Segments of mitochondrial DNA containing the structural genes of cytochrome oxidase have been isolated from rho minus clones of Saccharomyces cerevisiae. Detailed restriction maps of the DNAs have been constructed. The DNA segment of the clone DS67 has been used to sequence the oxil locus and the genes of methione and asparagine transfer RNAs. The gene product of the oxil locus has been identified to be subunit 2 of cytochrome oxidase. The DNA sequence of the subunit 2 gene has revealed that mitochondria make use of some unusual codons. Partial sequence data has also been obtained for the oxi 2 locus that is currently thought to code for subunit 3 of cytochrome oxidase.